Similar to other eukaryotes, the majority of mitochondrial proteins in Trypanosoma brucei, the infectious agent of a fatal disease (African trypanosomiasis) in human and domestic animals, are nuclear-encoded and need to be imported into mitochondria. However, the protein import machinery remains elusive in this organism. Fungi and higher eukaryotes possess three homologous proteins Tim17, Tim23, and Tim22, which formed two translocases (TIMs) in the inner membranes of mitochondria that have distinct specificities for different types of signal bearing proteins. In contrast, T. brucei possesses a single homolog of these proteins, TbTim17, which we found is present in a larger molecular mass protein complex in the mitochondrial inner membrane and is required for the import of both N-terminal and internal targeting signal containing proteins into mitochondria. Thus T. brucei may possess a single TIM complex capable of importing multiple types of matrix and inner membrane proteins. How a single protein in T. brucei performs functions of three different proteins in other eukaryotes remains enigmatic. We also have identified a Tim17- interacting protein (TbTim50) and genome searches indicate that there are other Tim50 related proteins in T. brucei. All of these variants possess a characteristic phosphatase domain. In addition to its role in mitochondrial protein import and cell growth, we found that TbTim50 over expression reduces mitochondrial ATP production via regulation of the expression level of the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. Together, this suggests that mitochondrial protein importer molecules such as Tim50 may be linked to other activities of this organelle besides importing proteins into mitochondrial matrix and inner membrane. In this project we will characterize the function of Tim50 and Tim50-related proteins in T. brucei and identify the Tim17 and Tim50 interacting domain(s) crucial for their function. We will also investigate how Tim50 is connected to VDAC expression, which is connected to regulation of mitochondrial metabolic activities. We will use various gene manipulation techniques, in vitro and in vivo protein import assays, FRET analysis, yeast two-hybrid analysis, and expression of TbTim50 mutants to study the function and protein/protein interactions of Tim50 and Tim17 in T. brucei. Characterization of the novel structure and function of T. brucei Tims may provide insights that can be utilized to define a new chemotherapeutic target for African trypanosomiasis.